This invention relates to a process for the extraction, separation and recovery of diterpene glycosides naturally occurring in the plant Stevia rebaudiana Bert. Hemsl. (Compositae), herein referred to as S. rebaudiana.
The desire for low calorie, or no-calorie, sweeteners led originally to the use of artificial sweeteners such as the cyclamates and saccharin, as substitutes for sucrose. Questions of their effects on health having arisen, a search for other non-sucrose sweeteners is in progress. The search has turned to sweeteners of natural origin, the rationale being that they would be less likely to have harmful effects. Many natural sweeteners, however, have disadvantages relating to the taste sensations they produce, such as a low degree of sweetness or an unpleasant aftertaste. Other disadvantages may include decomposition when heated during cooking.
S. rebaudiana, a plant native to Paraguay, has been used as a sweetening agent. The leaves of this plant have been reported to contain at least eight structurally related diterpene glycosides (referred to hereinafter at times as DTG materials): steviolbioside, stevioside, rebaudiosides A to E and dulcoside A. Table 1 depicts their structural formulae. A number of these glycosides are sweet in taste. Stevioside, rebaudioside A and rebaudioside C (dulcoside C) are present in the largest quantities and are the sweetest of the DTG materials. Efforts have been made to recover and separate the sweetest components for commercial use as sweeteners. U.S. Pat. No. 4,082,858, issued to Morita et al., describes a process of separation and isolation of one of the sweet-tasting diterpene glycosides (rebaudioside A).
TABLE 1 ______________________________________ ##STR1## COMPOUND R.sub.1 R.sub.2 ______________________________________ Steviolbioside H G.sup.2.sup.1 G Stevioside G G.sup.2.sup.1 G Rebaudioside A G ##STR2## Rebaudioside B H ##STR3## Rebaudioside C (Dulcoside B) G ##STR4## Rebaudioside D G.sup.2.sup.1 G ##STR5## Rebaudioside E G.sup.2.sup.1 G G.sup.2.sup.1 G Dulcoside A G G.sup.2.sup.1 Rh ______________________________________ G = glucose Rh = rhamnose
Generally, the prior recovery processes involve:
1. Extraction of the S. rebaudiana plant material with a highly polar solvent such as water or methanol to give a DTG material-containing extract. PA0 2. Introduction of the DTG material, in a form suitable for liquid chromatography, onto a liquid chromatography column, elution with a polar solvent, and identification and separation of the eluate fraction(s) containing DTG material. PA0 3. Removal of the eluting solvent from the fraction(s) containing DTG material.
Ordinary liquid chromatography imposes trade-offs between the factors of purity of product and quantity of product purified in a given time: the higher the purity the less the quantity of product purified per unit of time. While very large columns have been used commercially to obtain high purity at a reasonable output of product, it has been found previously that such results may be obtained in a more compact system, employing high performance liquid chromatography (HPLC) equipment, which uses high fluid pressures to drive eluting solvent continuously through very tightly-packed high surface area packing. Thus, Y. Hashimoto and his coworkers reported (Shoyakugaku Zasshi 32:209 [1978]; J. Chromatography 161:403 [1978]) the separation of two of the S. rebaudiana DTG materials, stevioside and rebaudioside A, with an HPLC system. So far as we are aware, however, no prior single liquid chromatographic system has been reported to separate all of the eight DTG materials known to be recoverable from S. rebaudiana. It would be desirable to effect such separation with a single system and in a unitary operation.